Abstract

In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Δyar1 mutants, but does not suppress either defect in Δltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.

ALL eukaryotic cells share the ability to sense and respond to a wide variety of environmental stress conditions. In the yeast Saccharomyces cerevisiae, screens for mutants hypersensitive to specific stress conditions have identified genes required for the detection and signaling of stress, as well as genes with functions in repair of stress-induced damage or in the establishment of a stress-tolerant state (Jamieson 1998; Estruch 2000; Hohmann 2002). More recently, genomic expression-profiling studies have revealed that a wide variety of stress conditions, ranging from starvation, heat shock, oxidization, or osmotic stress, all induce a large, stereotypical remodeling of gene expression (Gaschet al. 2000; Caustonet al. 2001). About two-thirds of the ∼900 genes included in this genomic “environmental stress response” are transiently repressed; the rest are transiently induced. Of the genes induced by multiple stresses, many have known functions in the stress response. However, not all genes that are induced/repressed in common by stress are thought to function directly to protect the cell from stress. Rather, many of the changes in gene expression may represent an adaptive adjustment in cellular metabolism under nonoptimal growth conditions.

One cluster of genes whose expression is coordinately and transiently repressed by multiple environmental stresses is composed almost entirely of genes that encode proteins with functions in ribosome structure, function, or biogenesis (Gaschet al. 2000; Caustonet al. 2001). The ribosome is an enormous machine, assembled from four rRNAs and 80 ribosomal proteins. Ribosome biogenesis takes place primarily in the nucleolus where transcription of ∼200 tandemly repeated rDNA genes produces 35S precursor rRNAs. The 35S pre-rRNA assembles with both ribosomal and nonribosomal proteins to form a 90S preribosome complex, which is subsequently processed into 66S and 43S preribosomal subunits. Further cleavage of the rRNA and maturation of the 66S preribosomes takes place in the nucleolus and nucleoplasm, with the final maturation of the 43S particle occurring in the cytoplasm (Kressleret al. 1999; Venema and Tollervey 1999; Fatica and Tollervey 2002; Fromont-Racineet al. 2003). Exponentially dividing yeast cells have been estimated to produce new ribosomes at a rate of almost 40/sec (Warner 1999). This makes ribosome synthesis a major cellular biosynthetic activity, and the coordinate repression of the expression of these ribosomal components under adverse environmental conditions would liberate significant energy resources for other cellular processes.

We previously identified YAR1 as a small gene required for a normal rate of proliferation, whose transcription is strongly and transiently repressed by heat shock (Lycanet al. 1996). Yar1p is composed almost entirely of two ankyrin repeats, which are conserved 33-amino-acid motifs that occur in tandem and fold to form L-shaped protein:protein interaction domains (Sedgwick and Smerdon 1999). While most ankyrin-repeat-containing proteins are large multidomain proteins with diverse cellular functions (Bork 1993), a more limited group of small ankyrin proteins, composed mostly or entirely of ankyrin repeats, functions simply by binding and hence regulating their nonankyrin partners. For example, the ankyrin-repeat protein IkBα regulates the cellular location of transcription factor NFkB by masking its nuclear localization signal (NLS) (Vermaet al. 1995), while pINK4 inhibits the enzymatic activity of its partner, CDK4/6 (Serranoet al. 1993; Hannon and Beach 1994; Chanet al. 1995; Hiraiet al. 1995; Guanet al. 1996). In this study, we identify a novel role for a small ankyrin-repeat protein in ribosome biogenesis. We provide genetic and biochemical evidence that Yar1 physically interacts with ribosomal protein S3 and with Ltv1, a protein recently copurified with a number of proteins implicated in 43S preribosome processing (Schaferet al. 2003). We demonstrate that both Δyar1 and Δltv1 mutants are hypersensitive to certain protein synthesis inhibitors and exhibit aberrant polysome profiles with a reduced absolute number of 40S subunits and an excess of free 60S subunits, relative to wild-type cells. In addition, both mutants are hypersensitive to osmotic and oxidative stress, as well as to low- and high-temperature conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Δyar1 mutants, but not that of Δltv1 mutants. On the basis of these and other results, we propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress.

MATERIALS AND METHODS

Yeast manipulation:

Yeast were cultured and manipulated according to standard laboratory practices, which have been described previously (Guthrie and Fink 1991). Plates for stress and protein synthesis inhibitor assays were made by adding each drug (Sigma, St. Louis), dissolved in water (except anisomycin, which was dissolved in ethanol), to YPD agar cooled to 50°–55°, to the final concentration noted. Sorbitol plates were made by adding sorbitol to 1.5 m prior to autoclaving. Yeast transformations were performed using the lithium acetate method (Gietzet al. 1992).

All yeast strains used in this study are listed in Table 1. LY103 was constructed by one-step disruption of the YAR1 locus by transformation of W303 with Ld6 (Lycanet al. 1996) cleaved with ClaI and KpnI; disruption of YAR1 was confirmed by Southern blot analysis. The heterozygous diploid was sporulated to produce LY103, -104, -105, and -106, four haploid segregants of one tetrad. LY124 was constructed by disruption of one RPS3 allele by transformation of LY101 (Lycanet al. 1996) with pSK2 (Finken-Eigenet al. 1996) digested with XbaI and XhoI; the presence of one wild-type and one rps3::HIS3 allele was confirmed by PCR. LY126 was generated similarly, except that W303 was used for transformation with pSK2. LY141 was constructed by disrupting the YAR1 locus in LY139 with Ld6 cleaved with ClaI and KpnI (confirmed by PCR). We sporulated this heterozygous diploid and the Δyar1::URA3 allele segregated 2:2 in seven tetrads. LY141 is a G418-resistant, Ura+ haploid segregant from one of these seven tetrads. Strains LY150 and LY156 were generated by transforming W303 haploids with PCR-amplified pFA6a-13Myc-His3MX6 and pFA6a-3HA-kanMX6, respectively, as described (Longtineet al. 1998). Integration of the tag at the genomic locus in each strain was confirmed by PCR and production of the correct fusion protein was confirmed by Western blot analysis. LY161 was generated by mating LY150 and LY156, sporulating the diploid, and selecting for both G418 resistance and histidine prototrophy in random spores. LY161 was confirmed to be haploid by mating tests.

Construction of plasmids:

We constructed pYAR1cen (Ld25) by amplifying the intergenic region between YAR1 and HSP82 using a forward primer that anneals upstream of HSE1 in the HSP82 promoter and a reverse primer that anneals within the YAR1 coding region. The PCR product was cleaved with BamHI and NdeI and ligated into pRS314 (Sikorski and Hieter 1989) containing the YAR1 ORF (Ld3). Plasmid structure was confirmed by restriction analysis. Transformation of LY103 with Ld25 confirmed that the centromere vector is able to fully complement the cold-sensitive, heat-sensitive, and slow-growth phenotypes of the Δyar1 strain.

To create pLexA-YAR1 (Ld17), the YAR1 ORF was amplified by PCR, ligated into pCRII-TA (Invitrogen, San Diego), and then subloned into SalI/PstI-digested pLEXA (gift of Stan Hollenberg). Ld17 retains the first codon for methionine of the YAR1 ORF and adds two codons (for amino acids Pro and Asp) between the SalI site and the YAR1 ATG. The in-frame LexA-YAR1 fusion junction was confirmed by DNA sequencing. Ld20 was derived from Ld17 by cleaving with AflII and religating the vector to delete 1223 bp and the NdeI site from the ADE2 gene. To create pGEX-YAR1 (Ld19), we subcloned YAR1 from Ld17 as a SalI/NotI fragment into pGEX-6P-1 (Pharmacia). The presence of an in-frame fusion between GST and YAR1 and the absence of any amino acid substitutions in the YAR1 ORF were confirmed by DNA sequencing.

RPS3 and LTV1 ORFs were each cloned into pCITE2a (Novagen) for in vitro transcription/translation as follows. The LTV1 ORF was amplified from genomic W303 DNA using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and PCR primers. The primers create an NcoI site that includes the initiator codon (Met) for the protein at the 5′ end and an AvaI site immediately upstream of the stop codon at the 3′ end. The PCR product was cleaved with NcoI and AvaI and ligated into pCITE2a cleaved with the same enzymes. This creates an S2A substitution at position 2 in the LTV1 sequence. The sequence of Ltv1 is otherwise unchanged except for the addition of LEHHHHHH at the C terminus. RPS3 was cloned into pCITE2a similarly, except pSK1 (gift of M. Finken-Eigen) was used as a template for PCR instead of genomic DNA, and the forward primer creates an NcoI site without altering the amino acid sequence of the protein. Thus the RPS3 sequence is fully wild type except for the addition of LEHHHHHH at the C terminus. The sequence of all constructs was confirmed to be wild type, except for the changes expected, by DNA sequencing.

Site-directed mutagenesis:

We mutated both K91 and K125 to alanine using a QuikChange site-directed mutagenesis kit following the protocol provided by the manufacturer (Stratagene). The mutagenic primer sets used to generate the K91A mutation were 5′-gttaatgaggtgaatgcaacaggcaacacggctttacattgggc-3′ and its complement, and for K125A, 5′-gcagacccctttattagaaacgcgttcggccacgatgc-3′ and its complement. Engineered BsmI and MluI restriction sites are italicized. The presence of the intended mutations and the absence of other mutations were confirmed by DNA sequencing.

Two-hybrid screen:

A two-hybrid screen for Yar1-interacting proteins was performed in yeast strain L40 harboring the bait plasmid pLexA-YAR1 (LD17). L40 pLexA-YAR1 was transformed with a yeast cDNA library fused to the Gal4 activation domain in pGAD24 (gift of S. Elledge). We screened 3.8 × 105 yeast Trp+ transformants for lacZ expression using standard protocols (Vojtek and Hollenberg 1995) and picked 175 positives, of which 162 retested positive in a second assay of purified colonies. All of these were identified to be one of three genes by direct sequencing or by screening with RPS3-specific primers. For all three genes, we showed that loss of the bait plasmid correlated with loss of lacZ transcriptional activation and that each of the activating plasmids could again confer the His+Lac+ phenotype when reintroduced into cells containing the pLexA:YAR1 bait plasmid.

β-Galactosidase activity assays:

β-Galactosidase activity was assayed as described (Breeden and Nasmyth 1985) and normalized to protein levels using the following equation: 1000 × (OD420/ml of supernatant)/protein concentration (mg/ml of supernatant) × reaction time (min). Individual single colonies were inoculated into 5 ml selective media and grown for 17 hr at 30° until the OD660 of the culture was between 0.7 and 1.3.

In vitro interaction assays:

GST pulldown assays were performed essentially as described (Ausubelet al. 1995). BL21 cells, transformed with the pGEX-6p-1 plasmid (Pharmacia) or with pGEX-YAR1, were induced for 2 hr with 0.4 mm isopropyl-1-thio-b-d-galactopyranoside (Fisher). Harvested cell pellets were lysed by sonication and cleared cell lysates (2 mg/ml total protein) were incubated with glutathione-linked agarose beads (Sigma) at room temperature for 5 min. The ratio of lysate volume to bead volume was between 5:1 and 10:1 in all experiments. After binding, beads were washed five times with five bed volumes of bead binding buffer.

Potential Yar1 partner proteins (cloned in pCITE2a vectors) were synthesized in vitro using an STP3 transcription/translation kit (Novagen) and 40 μCi [35S]methionine (Amersham Pharmacia) according to the manufacturer's specifications. Radiolabeled protein was incubated with GST or GST-YAR1-loaded glutathione-agarose beads in the presence of 1–2 mg of BL21 extract for 1 hr at 4°. GST and GST-YAR1 loaded beads were then washed three times and bound protein was eluted by boiling beads 5 min in 1× electrophoresis sample buffer. Eluted proteins were analyzed on 12% polyacrylamide gels. Proteins were visualized and quantified using phosphorimaging (Molecular Dynamics, Sunnyvale, CA).

High-copy suppressor screen:

Strain LY135 was transformed with a yeast centromere GAL1-regulated cDNA library (Liuet al. 1992; provided by L. Breeden) as described (Adamset al. 1998) with the following modifications: DMSO was added to 10% of the volume before the heat-shock step, and transformants were allowed to recover for 2 hr in YPD before being plated onto selective plates. We plated ∼35,000 transformants onto selective plates containing 2% galactose and 5 mg/ml neomycin. Transformants with suppressing plasmids were patched onto selective galactose/neomycin plates, replica printed onto plates containing either glucose or galactose and neomycin, and scored relative to LY135 and LY134 strains. Plasmids were rescued from transformants that grew better than Δyar1 on galactose only and retransformed back into LY135. Plasmids that again conferred galactose-dependent suppression were characterized by restriction endonuclease digestion and sequenced from the 3′ end to identify the insert. The sequence of both strands of one of the RPS3-containing suppressing plasmids was determined to confirm that the cDNA was full length and without mutations. This plasmid, pGAL:RPS3, was used for all subsequent analysis.

RESULTS

Yar1 interacts with RpS3 and Ltv1:

We previously reported that YAR1 encodes a small ankyrin-repeat protein. Deletion of the gene is not lethal, but leads to a slow-growth phenotype that is especially pronounced when cells are grown at low or high temperature (Lycanet al. 1996). To further characterize YAR1, we undertook a yeast two-hybrid screen to identify interacting protein partners. YAR1, fused to the LexA DNA-binding domain, was the bait in a screen of a yeast cDNA library. Of 162 transformants that retested positive in a second assay, 158 were shown to contain RPS3, which encodes ribosomal protein S3, a component of the small subunit of the ribosome. Two other genes were recovered in this screen: LTV1 and an uncharacterized ORF, YOR021c. Each was recovered twice as independent isolates from the library. While we find genetic evidence in support of Yar1:RpS3 and Yar1:Ltv1 protein:protein interactions (see below), we were not able to detect either an in vitro interaction between Yar1 and YOR021c or any genetic evidence in support of this interaction, and so this gene was not characterized further. RpS3 is a highly conserved, ribosomal protein that is part of the 40S subunit, with proposed roles in translation initiation (Westermannet al. 1981), decoding accuracy (Hendricket al. 2001), and repair of oxidative DNA damage (Kimet al. 1995; Yacoubet al. 1996; Deutschet al. 1997; Sandigurskyet al. 1997). Ltv1 is a conserved protein of unknown function that was recently identified as a component of a purified preribosomal complex (Schaferet al. 2003).

To determine whether any of the YAR1-interacting proteins detected in our two-hybrid screen also interact with Yar1 in vitro, we cloned YAR1 as an N-terminal GST fusion and expressed it in Escherichia coli. Each interacting protein was synthesized and labeled in vitro and then tested for retention by GST-YAR1-loaded glutathione agarose beads. Since the interacting proteins are synthesized in mammalian cell extracts, retention of labeled protein by GST-YAR1 is evidence that the interaction does not require other yeast proteins. Labeled RpS3 was specifically retained by the GST-YAR1 loaded beads and not by GST-loaded beads (Figure 1A). Quantitative analysis of the phosphorimager screen images indicate that ∼10% of the input labeled RpS3 protein was retained by GST-YAR1, compared to 0.2% by the GST beads. In contrast, no significant amount of labeled Ltv1 was retained by the GST-YAR1 beads over the levels retained nonspecifically by the GST beads alone (Figure 1B). These data indicate that the interaction between Yar1 and RpS3 can occur in the absence of other yeast proteins but that between Yar1 and Ltv1 must be indirect.

Yar1 interacts with RpS3 in vitro. GST and GST-Yar1 expression was induced in BL21 cells and lysates incubated with glutathione agarose beads. (A) RpS3 (transcribed and translated in vitro in rabbit reticulocyte lysates in the presence of [35S]methionine) was incubated with an excess of E. coli cell lysate and then added to glutathione agarose beads previously loaded with 4 μg of GST or GST-Yar1 protein. After extensive washing, the beads were boiled in 1× sample buffer and the supernatant analyzed by SDS-PAGE. Input lanes contain 5% of the reaction loaded onto the beads; bead lanes contain 90% of the supernatant boiled off the beads. (B) Ltv1, expressed and labeled as above, was incubated with GST- and GST-Yar1-loaded glutathione agarose beads as described in A.

The Yar1 ankyrin repeats are required for the two-hybrid interaction between Yar1 and Ltv1:

The co-crystal structures of a number of ankyrin-repeat proteins and their nonankyrin partners have identified variable residues at the tips of the β-hairpin loops of the ankyrin motif as frequent sites of specific protein:protein interaction (Gorina and Pavletich 1996; Brothertonet al. 1998; Russoet al. 1998). We aligned the amino acid sequence of Yar1 with the known structure of the CDK inhibitor, INK4p18. The optimal alignment of Yar1 is with the third and fourth ankyrin repeats of INK4p18 (Figure 2A). This alignment predicts that each β-hairpin loop in Yar1 has a positively charged lysine residue at the tip (highlighted in yellow).

The β-hairpin lysines in the ankyrin domain are critical for the Yar1:Ltv1 interaction. (A) The Cn3D program at NCBI was used to map the aligned (by gapped BLAST) amino acid sequence of the Yar1 ankyrin domain onto the 3D crystal structure of INK4p18. The program optimally aligned the Yar1 repeats with the third and fourth ankyrin repeats of INK4p18. In this representation, red represents identical residues; blue, nonconserved residues; and purple, conservative substitutions. The two positively charged lysines at the tip of each Yar1 loop have been highlighted in yellow. (B and C) Two-hybrid strains containing wild-type (WT Yar1) or mutant forms of Yar1 (K91A, K125A, or K91,125A), fused in frame with the LexA-DB and the Ltv1- or the RpS3-activation domain fusion plasmids, were grown at 30°. Three individual colonies were inoculated in each case and cell pellets were analyzed for β-galactosidase activity and protein content. Values shown represent the mean of three separate assays, with error bars representing 1 SD of the data.

We changed each of these lysines to alanine, both individually and in combination, by site-directed in vitro mutagenesis of YAR1 in the pLexA:YAR1 bait plasmid. These plasmids were then reintroduced into yeast carrying either RPS3 or LTV1 as activation-domain fusion plasmids. The resulting transformants were assayed for β-galactosidase activity as an indirect measure of their interaction. A representative experiment is shown in Figure 2, B and C. Mutation of lysine 91 reduces, and mutation of both lysines, eliminates the interaction between Yar1 and Ltv1 (Figure 2B). These data indicate that the tips of the ankyrin repeats are critical for the Yar1:Ltv1 two-hybrid interaction. Mutation of both lysines at the β-hairpin tips has essentially no effect on the interaction between Yar1 and RpS3 (Figure 2C). This suggests that Yar1 interacts with RpS3 and Ltv1 through different surfaces of the protein.

Stress-sensitive phenotypes of Δyar1 and Δltv1 mutants:

We previously reported that Δyar1 strains are viable but slow growing under either heat-shock (37°) or low temperature (25°) growth conditions (Lycanet al. 1996). As incubation at such temperatures is stressful for yeast, we asked whether Δyar1 cells exhibited any other stress-sensitive phenotypes. Specifically, we tested Δyar1 cells for sensitivity to oxidative stress, high osmolarity, and nutrient and amino acid starvation. Cells lacking YAR1 were sensitive to oxidative stress induced by treatment with diamide, at least as sensitive as cells lacking YAP1, the major oxidative stress-activated transcription factor (Jamieson 1998; see Figure 3A). Diamide acts to deplete glutathione pools and oxidizes thiol groups (Kosower and Kosower 1987). The Δyar1 strain was also sensitive to osmotic stress induced by sorbitol, although not as sensitive as the Δpbs2 mutant. PBS2 encodes the MAPKK that phosphorylates Hog1 in response to high-salt or sorbitol growth conditions (Hohmann 2002). Thus YAR1 is required for optimal long-term growth under both oxidative and osmotic stress conditions. However, the Δyar1 mutant showed no defects in viability after prolonged nitrogen deprivation (9 days). Mutant cells also accumulated storage carbohydrates comparable to wild-type cells, and the sporulation efficiency was essentially the same as in wild-type controls (data not shown). We also tested the phenotype of Δyar1 mutants constructed in the S288c background, in addition to our W303 background. Δyar1 cells exhibit the same phenotypes in both strain backgrounds (Figure 3B).

Δyar1 strains and Δltv1 cells exhibit stress-sensitive phenotypes. (A) Wild-type parental strain W303 and isogenic deletion derivatives Δyar1::URA3 and Δpbs::LEU2 were grown to midlog phase in YPD media, as were parental strain SEY6210 and its isogenic deletion derivative, Δyap1-Δ2. Cell density was determined by hemocytometer and 5-μl aliquots containing 16,000 (left) and 4000 (right) cells were spotted onto YPD or YPD plus the indicated drugs. In this experiment, 1.3 times more Δyar1 and Δpbs2 cells were spotted at each position compared to the other strains to compensate for the slightly slower growth rate of these mutants under nonstress conditions. Plates were incubated at 30° and photographed after 2 days. The W303 strain background is more sensitive than the SEY6210 strain to diamide. (B) Parental strain LY134 (derived from S288c) and isogenic deletion derivatives LY135 and LY136 were grown as in A. Equal numbers of wild-type and mutant cells were spotted at each position. All plates were photographed after 2 days at 30° except the 18° plate, which was photographed after 3 days of incubation.

If Yar1 and Ltv1 physically interact in vivo, then we would predict that strains lacking either protein might share one or more phenotypes. We therefore asked whether strains lacking LTV1 shared any of the stress-sensitive phenotypes of Δyar1 cells. We found that the Δltv1 strain is hypersensitive to both osmotic and oxidative stress and is especially cold sensitive (Figure 3B). However, while LTV1 stands for low-temperature viability, we find the Δltv1 strain is slow growing but viable at all of the low temperatures that we tested (18°, 15°, 12°, and 10°; doubling time at 18° is ∼7.5 hr). The Δltv1 strain, like the Δyar1 strain, has a slight slow-growth phenotype at 30°. The fact that Δyar1 and Δltv1 cells share multiple genetic phenotypes (as well as antibiotic hypersensitivity; see below) argues that the two-hybrid interaction between these two proteins reflects a common biological function in vivo.

Genetic interaction between YAR1 and RPS3:

Eukaryotic RpS3 is a highly conserved 36-kD protein located on the solvent side of the 40S subunit on the beak of the head region (Spahnet al. 2001). Multiple functions have been proposed for this small protein. Mammalian RpS3 can be crosslinked to eIF3 (Tolanet al. 1983) and eIF2 (Westermannet al. 1979), the basis for its proposed role in translation initiation (Westermannet al. 1981). In yeast, the missense allele of RPS3, suf14-1, is resistant to aminoglycosides and acts as an extragenic suppressor of +1 frameshift mutations, implicating RpS3 in translational decoding (Hendricket al. 2001). Finally, in vitro assays have implicated mammalian and Drosophila RpS3 in the repair of oxidative/UV DNA damage (Kimet al. 1995; Yacoubet al. 1996; Deutschet al. 1997; Sandigurskyet al. 1997), and yeast RpS3 has been shown to have an endonuclease activity on apurinic DNA in vitro (Junget al. 2001).

To test the biological function of the detected two-hybrid and in vitro interactions between RpS3 and Yar1, we turned to genetic analysis. One way to uncover a genetic interaction, when one of the two genes is essential, is to reduce the dosage of the essential protein in a background in which the nonessential protein is eliminated. We constructed a diploid strain, homozygous for Δyar1 and heterozygous for Δrps3, and tested it for a synthetic growth defect under various stress conditions. The heterozygous Δrps3/+ strain was indistinguishable from the wild-type parent under both optimal and all stress growth conditions that we tested (Figure 4a). The Δyar1/Δyar1 diploid is slow growing at 23° and at 37°, but is less affected by temperature than the haploid strain. However, the Δyar1/Δyar1, Δrps3/+ strain grew more slowly than either the Δyar1/Δyar1 diploid or the ΔrpS3/+ strain at all temperatures, but especially at 23° and 37° (Figure 4a). In addition, Δyar1/Δyar1, ΔrpS3/+ is more sensitive to osmotic stress and oxidative stress than either the Δyar1/Δyar1 strain or the ΔrpS3/+ strain (Figure 4b). The synthetic phenotype produced when the RPS3 dosage is reduced in the Δyar1/Δyar1 background is evidence of a genetic interaction between YAR1 and RPS3. These data are consistent with the evidence of a physical interaction between the two proteins in the two-hybrid screen and in vitro.

Genetic interaction between YAR1 and RPS3. (a) Diploid strains carrying the mutations indicated below were streaked for single colonies on YPD plates and incubated at 23°, 30°, or 37°. Homozygous Δyar1 diploids are not as sensitive to temperature stress as are the haploid strains. (A) +/+, Δyar1/Δyar1, (B) Δrps3/+, Δyar1/Δyar1, (C) +/+, +/+, and (D) Δrps3/+, +/+. (b) Diploid strains bearing the indicated mutations were grown as in Figure 1 and 16,000, 4000, and 1000 cells were spotted (left to right) onto YPD, YPD + diamide, and YPD + sorbitol plates. To compensate for the slower growth rate of the two homozygous Δyar1/Δyar1 strains on YPD, 1.3 times more cells were spotted at each position for these two strains relative to the others. Plates were incubated at 30° and photographed after 25 hr of incubation.

YAR1 and LTV1 mutants are hypersensitive to protein synthesis inhibitors:

Identification of RpS3 and Ltv1 as interacting partners for Yar1 led us to look for further evidence that might link Yar1 to ribosome structure or function in vivo. Strains with mutations in genes involved in ribosome function or biogenesis often exhibit an altered sensitivity to drugs that affect protein synthesis (Leeet al. 1992; Nelsonet al. 1992; Kressleret al. 1997; Yanet al. 1998; Liu and Thiele 2001). We tested four inhibitors of translational elongation. Aminoglycosides like paromomycin and neomycin bind to the A-site decoding region of the small subunit reducing translational accuracy (Carteret al. 2000; Ryudoet al. 2002; Lynchet al. 2003). Anisomycin binds the 28S rRNA in the 60S subunit and inhibits the peptidyl transferase reaction (Rodriguez-Fonsecaet al. 1995). Cycloheximide inhibits translation by abrogating the translocation of the peptidyl tRNA from the A-site to the P-site (Pestka 1971). Cells lacking YAR1 are hypersensitive to anisomycin, but are no more sensitive to cycloheximide than are wild-type cells (Figure 5B). They are also hypersensitive to both of the aminoglycosides tested, neomycin and paromomycin (Figure 5A). Strains lacking LTV1 were sensitive to the same spectrum of protein synthesis inhibitors as are Δyar1 cells (Figure 5). These data further link Yar1 and Ltv1 function and suggest that both proteins have a role in ribosome biogenesis or function.

Both Δyar1 and Δltv1 strains are hypersensitive to protein synthesis inhibitors. The sensitivity of wild-type (WT) and isogenic Δyar1 and Δltv1 cells to various protein synthesis inhibitors was tested by growing cells as described in Figure 1. (A) A total of 16,000, 4000, or 1000 cells (left to right) were spotted onto YPD plates with or without the denoted drugs. Only the 1000-cell column is shown for the neomycin (neo; 1 mg/ml) and paromomycin (paro; 1 mg/ml) plates. (B) A total of 400 cells of each strain were spotted onto YPD, YPD + cycloheximide (CHX; 1μg/ml), or YPD + anisomycin (Anis; 7.5 μg/ml) plates. Equal numbers of wild-type and mutant cells were spotted at each dilution. Plates were incubated at 30° unless otherwise indicated.

Yar1 and Ltv1 are required for normal ribosome biogenesis:

Both biochemical interactions and mutant phenotypes link Yar1 and Ltv1 to RpS3 and to the ribosome. To test whether ribosome biogenesis is impaired in mutants lacking either of these proteins, we profiled cellular ribosomes from wild-type, Δyar1, and Δltv1 strains on sucrose density gradients. As shown in Figure 6, cells lacking YAR1 or LTV1 have aberrant polysome profiles relative to wild-type cells. Both mutants have a significantly enlarged 60S peak relative to isogenic wild-type cells. In addition, the total number of 40S subunits, determined by dissociating monosomes and polyribosomes into free subunits, is significantly diminished in the mutants; the 40S:60S ratio is reduced by 52% in Δyar1 and by 58% in Δltv1 (see insets in Figure 6). The reduction in total 40S subunits in both mutants is consistent with the excess of free 60S subunits in the polysome profile, and both pieces of data are consistent with a defect in 40S subunit biogenesis/maturation in both Δyar1 and Δltv1 mutants.

Strains lacking YAR1 and LTV1 have altered polysome profiles. Strains containing chromosomal deletions of YAR1 (LY135) and LTV1 (LY136) and their isogenic wild-type parental strains were grown at 30° in rich medium and then fractionated by mechanical disruption and centrifugation. The postnuclear cytosolic homogenate was separated on continuous sucrose gradients either containing 5 mm MgCl2 (full-size graphs) or lacking Mg2+ and containing 10 mm EDTA (insets). An RNA absorbance profile was determined using a continuous flow UV detector and is represented here in relative units. Only the portion of the gradient containing small subunits (40S), large subunits (60S), monomer ribosomes (80S), and small polysomes is shown. The arrow denotes the shoulder peak. The shaded line in the insets denotes the size of the wild-type small subunit peak.

One other aspect of the polysome profiles of Δyar1 and Δltv1 mutants is worth noting. In both mutant profiles, there is a distinct shoulder on the 80S peak. This shoulder peak disappears if extracts are centrifuged under conditions that dissociate monosomes and polysomes into free 40S and 60S subunits. Similar shoulder peaks, called “half-mer” peaks, have been noted in numerous 60S subunit biogenesis mutants and in mutants with defects in subunit joining (Woolford and Warner 1991; Kressleret al. 1999) and are composed, in these mutants, of 80S monosomes plus a stalled 43S preinitiation complex attached to the same mRNA. However, in 60S mutants, there are also “half-mer” peaks associated with each of the polysome peaks, which we did not observe. In addition, gel analysis of RNA extracted from the shoulder peak fraction reveals that it is enriched for large subunit rRNA (the 25S:18S ratio in the shoulder peak is 2.8 compared to a ratio of 1.7 in the 80S peak). This ratio suggests an excess of 60S subunits, not an excess of small subunits. Since both the Δyar1 and Δltv1 mutants have a deficiency in 40S subunits and an excess of free 60S subunits, the shoulder peak in these mutants is most likely dimers of free 60S subunits that form in the absence of sufficient 40S partners.

Ltv1 comigrates with ribosome subunits:

The observed defects in 40S subunit biogenesis in Δyar1 and Δltv1 mutants suggested that the interaction among Yar1 and Ltv1 and/or RpS3 might occur in the process of ribosome assembly. If so, we might expect Yar1 or Ltv1 to cosediment with ribosomes or preribosomal particles in vivo. To test this hypothesis, we epitope tagged each protein by integration of the epitope sequence at the appropriate chromosomal locus in different strains. Correct integration and synthesis of the fusion protein were confirmed in each strain by PCR and Western blot analysis. To generate a single strain (LY161) expressing both tagged proteins, we crossed the Yar1-HA strain to the Ltv1-Myc strain, sporulated the diploid, and selected for haploids with both fusion genes. We assessed the ability of each fusion protein to replace the function of the wild-type protein by testing these strains for slow growth and for stress sensitivity relative to wild-type and Δyar1 or Δltv1 strains. The Yar1-HA and the Ltv1-Myc strains, as well as the LY161 strain with both tagged proteins, grew as well as wild-type cells at 30° and on plates containing sorbitol, indicating that the tag does not interfere with the function of either protein (data not shown).

The association of Yar1 and/or Ltv1 with ribosomal particles was assessed by sucrose gradient centrifugation and Western blotting (polysome blots) of extracts of the Yar1-HA Ltv1-Myc (LY161) strain. When whole-cell lysates are prepared under low-salt conditions, most of the Yar1-HA is present in the cytosolic fractions, although some Yar1 is also detected in denser fractions of the gradient in the 40S region (Figure 7). The opposite is true of Ltv1; while some Ltv1-Myc is detected at the top of the gradient, the majority comigrates with 40S ribosomal subunits (Figure 7). Intriguingly, Ltv1 does not comigrate with polysomes. This is in contrast to RpS2, which, as expected for a structural component of the mature small subunit, comigrates with both 40S particles and with translating ribosomes. These data suggest that Ltv1 associates with the 40S subunit, but is released some time prior to its incorporation into a translating ribosome.

Association of Yar1p and Ltv1p with ribosomal subunits. Wild-type cells expressing epitope-tagged Yar1 and Ltv1 fusion proteins (LY161) were cultured at 30° and fractionated to yield a postnuclear cytosolic homogenate. This was separated on a continuous sucrose gradient as in Figure 6. Individual gradient fractions were processed for SDS gel electrophoresis and Western blotting with antibodies to small (S2) and large (L3) ribosomal subunit proteins and the myc and hemagglutinin (HA) epitopes.

RPS3 is a high-copy suppressor of the Δyar1 phenotypes:

To identify other genes acting in the same pathway as YAR1, we carried out a screen for high-copy suppressors of the neomycin-sensitive (neoS) phenotype of Δyar1 cells (see materialsandmethods). We characterized 95 suppressors that exhibited galactose-dependent suppression of the neoS phenotype, almost one-third of which turned out to be RPS3. RPS3 was isolated 31 times in this screen and confirmed to be a full-length cDNA without mutations. This result supports the genetic interaction noted earlier between YAR1 and RPS3 and indicates that the translation defect in Δyar1 cells that is detected by aminoglycoside hypersensitivity can be alleviated by the overexpression of this ribosomal protein. This result places the function of the YAR1 gene upstream of RPS3.

If the sensitivity of Δyar1 cells to protein synthesis inhibitors is a consequence of the defects noted in the 40S subunit assembly observed in mutant cells, then overexpression of RpS3 might be expected to alleviate the altered polysome profiles of Δyar1 mutants as well. To test this, we grew Δyar1 pGAL:RPS3 cells in glucose or galactose selective media and analyzed the two cell lysates by sucrose gradient centrifugation. As shown in Figure 8A, Δyar1 cells grown on glucose exhibit a characteristically enlarged 60S peak and a large shoulder on the 80S monomer peak. Both of these aberrant peaks are missing in cells grown on galactose. Thus overexpression of RPS3 suppresses both the neomycin sensitivity and the ribosome biogenesis defect of Δyar1 cells.

Overexpression of RPS3 suppresses the phenotypes of Δyar1 mutants. (A) Wild-type (LY134) cells transformed with pRS316 (Sikorski and Hieter 1989) and Δyar1 (LY135) and Δltv1 (LY136) cells transformed with pGAL:RPS3 were grown overnight at 30° in selective media including glucose and harvested by centrifugation. Half of the cells were resuspended in the same media, and half were resuspended in selective media including galactose. After a 7-hr incubation at 30°, cells were fractionated and the homogenate was centrifuged on continuous sucrose gradients, as described in Figure 6. The portion of the gradient containing small subunits (40S), large subunits (60S), monomer ribosomes (80S), and small polysomes is shown. Shaded lines indicate the glucose-grown samples, and solid lines indicate galactose-grown samples. (B) Δyar1 (LY135) cells transformed with pGAL:RPS3 or with pRS316 and wild-type (LY134) cells transformed with pRS316 were grown in selective medium to early log phase (OD660 0.2–0.7). Equal numbers of each strain were plated in fourfold dilutions (left to right) on selective plates containing either glucose or galactose and neomycin (5 mg/ml) or diamide (1 mm). Plates were grown at 30° for 2–5 days and photographed. (C) Cells were grown and diluted as described in B except that Δltv1 (LY136) cells transformed with pGAL:RPS3 or pRS316 were used instead of Δyar1 cells.

Cells lacking Yar1 are also hypersensitive to a number of environmental stress conditions. To explore further the link between the ribosome defects of Δyar1 mutants and their stress sensitivity, we tested whether overexpression of RPS3 on galactose could suppress the sensitivity of Δyar1 cells to oxidative stress (Figure 8B) or osmotic stress (data not shown). Cells transformed with pGAL:RPS3 grew as well as wild-type cells under all conditions. Suppression of the sensitivity in all cases was dependent on the presence of galactose. This surprising result suggests that it is unlikely that the multiple phenotypes of Δyar1 cells are due to multiple functions of Yar1. Rather, the ribosome defects and the stress-sensitive phenotypes are more likely directly linked, since overexpression of RPS3 can alleviate both.

Because Δyar1 and Δltv1 cells have very similar mutant phenotypes, we tested whether overexpression of pGAL:RPS3 could also suppress the stress-sensitive phenotypes of Δltv1. In contrast to Δyar1 cells, overexpression of the pGAL:RPS3 vector in Δltv1 mutants did not alleviate the sensitivity of this strain to either neomycin or oxidative stress (Figure 8C). Furthermore, overexpression of RPS3 did not suppress the polysome profile defect of Δltv1 (Figure 8A). We also tested whether overexpression of YAR1, in cells transformed with a pGAL:YAR1cen vector that we constructed, could suppress the Δltv1 stress-sensitive phenotypes. We did not observe any suppression (data not shown).

DISCUSSION

We characterize here a novel role for an ankyrin-repeat protein and uncover a putative new link between environmental stress and ribosome biogenesis. Yar1p interacts in a two-hybrid screen with two proteins associated with the small subunit of the ribosome, RpS3 and Ltv1. The Yar1:RpS3 interaction can be detected in vitro using proteins synthesized in E. coli or in mammalian cell extracts (Figure 1A), indicating that no other yeast proteins are required to mediate this interaction. While this suggests that the interaction is direct, we cannot exclude the possibility that a protein in the rabbit reticulocyte lysate used to synthesize RpS3 could be facilitating the interaction between Yar1 and RpS3. The biological relevance of the Yar1:RpS3 interaction is supported by evidence of genetic interaction. Reducing the RPS3 gene dosage in diploids lacking both YAR1 genes results in an enhanced stress-sensitive phenotype relative to Δyar1/Δyar1 diploids or to heterozygous ΔrpS3/+ mutants (Figure 4). Furthermore, RPS3 is a high-copy suppressor of both the neomycin hypersensitivity and the environmental stress sensitivity of Δyar1 cells (Figure 8).

The evidence that Ltv1 likely functions with Yar1 and RpS3 is several-fold. First, a two-hybrid interaction between Ltv1 and RpS3 has been reported by others (Itoet al. 2001), strengthening the link among these three proteins. Second, Δyar1 and Δltv1 cells exhibit an almost identical spectrum of stress-sensitive phenotypes (Figure 3). Third, Δltv1 is hypersensitive to the same spectrum of protein synthesis inhibitors as Δyar1 (Figure 5). This links both YAR1 and LTV1 to ribosome function/assembly.

Both Δyar1 and Δltv1 mutants exhibit clear defects in 40S-specific subunit production. The absolute number of 40S subunits is reduced in both mutants and the number of free 60S subunits is increased; the ratio of 40S:60S subunits declined by ∼50% in both strains relative to wild-type strains. This decrease in the 40S:60S ratio is consistent with the fact that these deletion strains are viable. Two other viable ribosome biogenesis deletion mutants, Δmrt4 and Δloc1, with defects in 60S subunit biogenesis, exhibit a similar (25–40%) decrease in the subunit ratio, in this case the 60S:40S ratio (Harnpicharnchaiet al. 2001). The increase in free 60S subunits observed in Δyar1 and Δltv1 cells has also been described for other 40S biogenesis mutants (Leeet al. 1992; Demianovaet al. 1996; Baudin-Baillieuet al. 1997; Liu and Thiele 2001; Milkereitet al. 2003; Tabb-Masseyet al. 2003) and has been ascribed to a paucity of functional small subunits with which large subunits may join. The hypersensitivity of Δltv1 and Δyar1 mutants to protein synthesis inhibitors, including aminoglycoside antibiotics (Figure 5), is most easily understood as a consequence of the reduction in the number of functional ribosomes in these mutants; hypersensitivity to aminoglycosides has been previously noted in numerous other ribosome mutants, including three that are specifically defective in 40S subunit biogenesis (Leeet al. 1992; Kressleret al. 1997; Liu and Thiele 2001).

Ltv1 is a conserved protein, which was recently identified as a minor component copurifying with 40S preribosomal complexes purified by tandem affinity purification tagging three different nonribosomal proteins (Schaferet al. 2003). The association of Ltv1 with pre-40S complexes is consistent with our identification of Ltv1 in polysome blots in the 40S region of the gradient. It is noteworthy that we do not detect Ltv1 in the 80S or polysome region of the sucrose gradient, which suggests that the protein is released from the 40S subunit at some point prior to the initiation of translation. Rio2, a nonribosomal protein that is required for maturation of a pre-40S particle, exhibits a cosedimentation profile essentially identical to that of Ltv1 (Vanrobayset al. 2003).

Strains lacking YAR1 exhibit the same ribosome biogenesis defects as cells lacking LTV1. Yar1 binds strongly with RpS3 in the two-hybrid and in vitro, yet it is only very weakly or transiently associated with 40S ribosomal subunits and has not been identified as a component of any preribosomal complex (Schaferet al. 2003). It may be that Yar1 interacts mostly with free RpS3, perhaps at a point prior to its incorporation into ribosomes. Since RPS3 overexpression can suppress the ribosome biogenesis defect of Δyar1 mutants, perhaps YAR1 functions as an RpS3 “chaperone,” regulating its stability, nuclear import, or incorporation into ribosomes in the nucleolus. Whether RpS3 itself has any direct role in ribosome assembly will require further investigation. However, it is increasingly clear that ribosomal proteins have multiple functions and may figure in other aspects of ribosome function in addition to translation itself. For example, the RpS0 proteins and RpS14 have been shown to be required for the maturation of the 40S subunit (Fordet al. 1999; Jakovljevicet al. 2004) and RpS15 is required for the nuclear exit of 40S preribosomal complexes (Leger-Silvestreet al. 2004).

Strains lacking either Yar1 or Ltv1 are stress sensitive, as well as exhibiting defects in ribosome biogenesis. Since ribosome biogenesis is closely regulated by nutrient availability and by environmental stresses (Warner 1999; Gaschet al. 2000), one hypothesis is that Yar1 and Ltv1 function to link ribosome biogenesis to stress-response signaling. The Yar1 protein seems especially well suited to a regulatory role, being composed essentially of a protein interaction domain with a strong potential PEST element in the C terminus and two potential casein kinase II recognition sites (Lycanet al. 1996). However, an alternative hypothesis is that the stress-sensitive phenotypes of Δyar1 and Δltv1 mutants are the consequence of a primary defect in ribosome biogenesis. At least for YAR1, the simplest interpretation of our data would support the latter interpretation. Overexpression of RPS3 in Δyar1 mutants suppresses both the ribosome biogenesis phenotype and the stress-sensitive phenotypes. This suggests that Yar1 does not have multiple functions, but that it primarily affects the expression or function of RpS3. Whether ribosomes made in the absence of Yar1 cause cells to be especially sensitive to stress conditions, or whether it is simply the reduction in the number of functioning ribosomes that makes cells stress sensitive is unknown as yet. Many cold-sensitive strains in E. coli have defects in ribosome subunit assembly (Guthrieet al. 1969) and numerous yeast strains with mutations in genes for ribosome proteins or trans-acting factors involved in rRNA processing are cold sensitive (Venema and Tollervey 1999). Likewise many mutants in ribosome function/biogenesis are aminoglycoside sensitive and slow-growing under normal conditions (Kressleret al. 1999). However, Δyar1 and Δltv1 strains display the additional phenotypes of sensitivity to osmotic and oxidative stress, which have not been reported for other ribosome biogenesis mutants. While viable knock-out strains with defects in ribosome biogenesis are rare, we did obtain two, Δmrt4 and Δloc1, with fewer functional ribosomes similar to Δyar1 and Δltv1, but in this case due to a reduction in 60S subunit production (Harnpicharnchai et al. 2001). Both these mutants are slow growing at 30° and cold sensitive and sensitive to neomycin, but only one of the two was sensitive to osmotic stress or oxidative stress (our unpublished observations). This suggests that the link between ribosome biogenesis and oxidative/osmotic stress sensitivity is at least complex and deserves further investigation.

Our results clearly indicate that Yar1 and Ltv1 play a role in both the biogenesis of 40S ribosomal subunits and the ability of yeast cells to adapt to various stress conditions. We are currently exploring the molecular basis of the link between these two phenotypes.

Acknowledgments

We acknowledge the following Lewis and Clark College alumni for their contributions to this project as undergraduates: Kimberley Stafford for the construction of pLEXA::YAR1 and Yuji Mishina for construction of the K91A YAR1 mutant. We thank B. Baxter for construction of the pCITE fusion vectors. We are grateful to Jim Posakony and Greg Hermann for reading the manuscript and for very helpful discussions. This work was supported by National Institutes of Health (NIH) grant GM-061643 to D.E.L. and by grant 97120:JVZ:02/19/98 from the M. J. Murdock Charitable Trust to D.E.L. In addition, R.M.S. was supported by NIH grant GM-068208.

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